A Deletion in the Endothelin-B Receptor Gene is Responsible for the Waardenburg Syndrome-Like Phenotypes of WS4 Mice.

The WS4 mouse is an animal model for human Waardenburg syndrome type 4 (WS4), showing pigmentation anomalies, deafness and megacolon, which are caused by defects of neural crest-derived cells. We have previously reported that the gene responsible for the WS4 mouse is an allele of the piebald mutations of the endothelin B receptor gene (Ednrb). In this study, we examined the genomic sequence of the Ednrb gene in WS4 mice and found a 598-bp deletion in the gene. The deleted region contains the entire region of exon 2 and the 5' part of exon 3 and is flanked by inverted repeat sequences which are suggested to trigger the deletion. We concluded that the deletion in the Ednrb gene is the causative mutation for the phenotype of WS4 mice.     

Partial requirement of endothelin receptor B in spiral ganglion neurons for postnatal development of hearing

Impairments of endothelin receptor B (Ednrb/EDNRB) cause the development of Waardenburg-Shah syndrome with congenital hearing loss, hypopigmentation, and megacolon disease in mice and humans. Hearing loss in Waardenburg-Shah syndrome has been thought to be caused by an Ednrb-mediated congenital defect of melanocytes in the stria vascularis (SV) of inner ears. Here we show that Ednrb expressed in spiral ganglion neurons (SGNs) in inner ears is required for postnatal development of hearing in mice. Ednrb protein was expressed in SGNs from WT mice on postnatal day 19 (P19), whereas it was undetectable in SGNs from WT mice on P3. Correspondingly, Ednrb homozygously deleted mice (Ednrb(-/-) mice) with congenital hearing loss showed degeneration of SGNs on P19 but not on P3. The congenital hearing loss involving neurodegeneration of SGNs as well as megacolon disease in Ednrb(-/-) mice were markedly improved by introducing an Ednrb transgene under control of the dopamine β-hydroxylase promoter (Ednrb(-/-);DBH-Ednrb mice) on P19. Neither defects of melanocytes nor hypopigmentation in the SV and skin in Ednrb(-/-) mice was rescued in the Ednrb(-/-);DBH-Ednrb mice. Thus, the results of this study indicate a novel role of Ednrb expressed in SGNs distinct from that in melanocytes in the SV contributing partially to postnatal hearing development.     

Cell-autonomous and cell non-autonomous signaling through endothelin receptor B during melanocyte development

The endothelin receptor B gene (Ednrb) encodes a G-protein-coupled receptor that is expressed in a variety of cell types and is specifically required for the development of neural crest-derived melanocytes and enteric ganglia. In humans, mutations in this gene are associated with Waardenburg-Shah syndrome, a disorder characterized by pigmentation defects, deafness and megacolon. To address the question of whether melanocyte development depends entirely on a cell-autonomous action of Ednrb, we performed a series of tissue recombination experiments in vitro, using neural crest cell cultures from mouse embryos carrying a novel Ednrb-null allele characterized by the insertion of a lacZ marker gene. The results show that Ednrb is not required for the generation of early neural crest-derived melanoblasts but is required for the expression of the differentiation marker tyrosinase. Tyrosinase expression can be rescued, however, by the addition of Ednrb wild-type neural tubes. These Ednrb wild-type neural tubes need not be capable of generating melanocytes themselves, but must be capable of providing KIT ligand, the cognate ligand for the tyrosine kinase receptor KIT. In fact, soluble KIT ligand is sufficient to induce tyrosinase expression in Ednrb-deficient cultures. Nevertheless, these tyrosinase-expressing, Ednrb-deficient cells do not develop to terminally differentiated, pigmented melanocytes. Pigmentation can be induced, however, by treatment with tetradecanoyl phorbol acetate, which mimics EDNRB signaling, but not by treatment with endothelin 1, which stimulates the paralogous receptor EDNRA. The results suggest that Ednrb plays a significant role during melanocyte differentiation and effects melanocyte development by both cell non-autonomous and cell-autonomous signaling mechanisms.     

Targeting of endothelin receptor-B to the neural crest

Endothelin receptor B (Ednrb) plays a critical role in the development of melanocytes and neurons and glia of the enteric nervous system. These distinct neural crest-derived cell types express Ednrb and share the property of intercalating into tissues, such as the intestine whose muscle precursor cells also express Ednrb. Such widespread Ednrb expression has been a significant obstacle in establishing precise roles for Ednrb in development. We describe here the production of an Ednrb allele floxed at exon 3 and its use in excising the receptor from mouse neural crest cells by use of Cre-recombinase driven by the Wnt1 promoter. Mice born with neural crest-specific excision of Ednrb possess aganglionic colon, lack trunk pigmentation, and die within 5 weeks due to megacolon. Ednrb receptor expression in these animals is absent only in the neural crest but present in surrounding smooth muscle cells. The absence of Ednrb from crest cells also results in a compensatory upregulation of Ednrb expression in other cells within the gut. We conclude that Ednrb loss only in neural crest cells is sufficient to produce the Hirschsprungs disease phenotype observed with genomic Ednrb mutations.     

Mouse models for four types of Waardenburg syndrome

Waardenburg syndrome (WS) is an auditory-pigmentary syndrome caused by a deficiency of melanocytes and other neural crest-derived cells. Depending on a variety of symptoms associated with the auditory-pigmentary symptoms, WS is classified into four types: WS type 1 (WS1), WS2, WS3, and WS4. Six genes contributing to this syndrome--PAX3, SOX10, MITF, SLUG, EDN3 and EDNRB--have been cloned so far, all of them necessary for normal development of melanocytes. Mutant mice with coat color anomalies were helpful in identifying these genes, although the phenotypes of these mice did not necessarily perfectly match those of the four types of WS. Here we describe mice with mutations of murine homologs of WS genes and verify their suitability as models for WS with special interest in the cochlear disorder. The mice include splotch (Sp), microphthalmia (mi), Slugh-/-, WS4, JF1, lethal-spotting (ls), and Dominant megacolon (Dom). The influence of genetic background on the phenotypes of mice mutated in homologs of WS genes is also addressed. Finally, possible interactions among the six WS gene products are discussed.     

Interactions between Sox10, Edn3 and Ednrb during enteric nervous system and melanocyte development

The requirement for SOX10 and endothelin-3/EDNRB signalling pathway during enteric nervous system (ENS) and melanocyte development, as well as their alterations in Waardenburg-Hirschsprung disease (hypopigmentation, deafness and absence of enteric ganglia) are well established. Here, we analysed the genetic interactions between these genes during ENS and melanocyte development. Through phenotype analysis of Sox10;Ednrb and Sox10;Edn3 double mutants, we show that a coordinate and balanced interaction between these molecules is required for normal ENS and melanocyte development. Indeed, double mutants present with a severe increase in white spotting, absence of melanocytes within the inner ear, and in the stria vascularis in particular, and more severe ENS defects. Moreover, we show that partial loss of Ednrb in Sox10 heterozygous mice impairs colonisation of the gut by enteric crest cells at all stages observed. However, compared to single mutants, we detected no apoptosis, cell proliferation or overall neuronal or glial differentiation defects in neural crest cells within the stomach of double mutants, but apoptosis was increased in vagal neural crest cells outside of the gut. These data will contribute to the understanding of the molecular basis of ENS, pigmentation and hearing defects observed in mouse mutants and patients carrying SOX10, EDN3 and EDNRB mutations.     

EDNRB/EDN3 and Hirschsprung disease type II.

The study of vertebrate pigmentary anomalies has greatly improved our understanding of melanocyte biology. One such disorder, Waardenburg syndrome (WS), is a mendelian trait characterized by hypopigmentation and sensorineural deafness. It is commonly subdivided into four types (WS1-4), defined by the presence or absence of additional symptoms. WS type 4 (WS4), or Shah-Waardenburg syndrome, is also known as Hirschsprung disease Type II (HSCR II) and is characterized by an absence of epidermal melanocytes and enteric ganglia. Mutations in the genes encoding the endothelin type-B receptor (EDNRB) and its physiological ligand endothelin 3 (EDN3) are now known to account for the majority of HSCR II patients. Null mutations in the mouse genes Ednrb and Edn3 have identified a key role for this pathway in the normal development of melanocytes and other neural crest-derived lineages. The pleiotropic effects of genes in this pathway, on melanocyte and enteric neuron development, have been clarified by the embryologic identification of their common neural crest (NC) ancestry. EDNRB and EDN3 are transiently expressed in crest-derived melanoblast and neuroblast precursors, and in the surrounding mesenchymal cells, respectively. The influence of EDNRB-mediated signaling on the emigration, migration, proliferation, and differentiation of melanocyte and enteric neuron precursors, in vivo and in vitro has recently been the subject of great scrutiny. A major emergent theme is that EDN3-induced signaling prevents the premature differentiation of melanocyte and enteric nervous system precursors and is essential between 10 and 12.5 days post-coitum. We review the present understanding of pigment cell development in the context of EDNRB/EDN3--a receptor-mediated pathway with pleiotropic effects.     

The endothelin receptor-B is required for the migration of neural crest-derived melanocyte and enteric neuron precursors

Mutations in the genes encoding endothelin receptor-B (Ednrb) and its ligand endothelin-3 (Edn3) affect the development of two neural crest-derived cell types, melanocytes and enteric neurons. EDNRB signaling is exclusively required between E10.5 and E12.5 during the migratory phase of melanoblast and enteric neuroblast development. To determine the fate of Ednrb-expressing cells during this critical period, we generated a strain of mice with the bacterial beta-galactosidase (lacZ) gene inserted downstream of the endogenous Ednrb promoter. The expression of the lacZ gene was detected in melanoblasts and precursors of the enteric neuron system (ENS), as well as other neural crest cells and nonneural crest-derived lineages. By comparing Ednrb(lacZ)/+ and Ednrb(lacZ)/Ednrb(lacZ) embryos, we determined that the Ednrb pathway is not required for the initial specification and dispersal of melanoblasts and ENS precursors from the neural crest progenitors. Rather, the EDNRB-mediated signaling is required for the terminal migration of melanoblasts and ENS precursors, and this pathway is not required for the survival of the migratory cells.     

Timeline and distribution of melanocyte precursors in the mouse heart

Apart from the well-studied melanocytes of the skin, eye and inner ear, another population has recently been described in the heart. In this study, we tracked cardiac melanoblasts using in situ hybridization with a dopachrome tautomerase (Dct) probe and Dct-LacZ transgenic mice. Large numbers of melanoblasts were found in the atrioventricular (AV) endocardial cushions at embryonic day (E) 14.5 and persisted in the AV valves into adulthood. The earliest time Dct-LacZ-positive cells were observed in the AV endocardial cushions was E12.5. Prior to that, between E10.5 and E11.5, small numbers of melanoblasts traveled between the post-otic area and third somite along the anterior and common cardinal veins and branchial arch arteries with other neural crest cells expressing CRABPI. Cardiac melanocytes were not found in the spotting mutants Ednrb s-l/s-l and Kit w-v/w-v, while large numbers were observed in transgenic mice that overexpress endothelin 3. These results indicate that cardiac melanocytes depend on the same signaling molecules known to be required for proper skin melanocyte development and may originate from the same precursor population. Cardiac melanocytes were not found in zebrafish or frog but were present in quail suggesting an association between cardiac melanocytes and four-chambered hearts.     

Mutational analysis of endothelin receptor b1 (rose) during neural crest and pigment pattern development in the zebrafish Danio rerio

Pigment patterns of fishes are a tractable system for studying the genetic and cellular bases for postembryonic phenotypes. In the zebrafish Danio rerio, neural crest-derived pigment cells generate different pigment patterns during different phases of the life cycle. Whereas early larvae exhibit simple stripes of melanocytes and silver iridophores in a background of yellow xanthophores, this pigment pattern is transformed at metamorphosis into that of the adult, comprising a series of dark melanocyte and iridophore stripes, alternating with light stripes of iridophores and xanthophores. Although several genes have been identified in D. rerio that contribute to the development of both early larval and adult pigment patterns, comparatively little is known about genes that are essential for pattern formation during just one or the other life cycle phase. In this study, we identify the gene responsible for the rose mutant phenotype in D. rerio. rose mutants have wild-type early larval pigment patterns, but fail to develop normal numbers of melanocytes and iridophores during pigment pattern metamorphosis and exhibit a disrupted pattern of these cells. We show that rose corresponds to endothelin receptor b1 (ednrb1), an orthologue of amniote Ednrb genes that have long been studied for their roles in neural crest and pigment cell development. Furthermore, we demonstrate that D. rerio ednrb1 is expressed both during pigment pattern metamorphosis and during embryogenesis, and cells of melanocyte, iridophore, and xanthophore lineages all express this gene. These analyses suggest a phylogenetic conservation of roles for Ednrb signaling in the development of amniote and teleost pigment cell precursors. As murine Ednrb is essential for the development of all neural crest derived melanocytes, and D. rerio ednrb1 is required only by a subset of adult melanocytes and iridophores, these analyses also reveal variation among vertebrates in the cellular requirements for Ednrb signaling, and suggest alternative models for the cellular and genetic bases of pigment pattern metamorphosis in D. rerio.     

Roles of endothelin signaling in melanocyte development and melanoma

Endothelin (Edn) signaling via the G-coupled, Edn receptor type B (Ednrb) is essential for the development of melanocytes from the neural crest (NC) and has been associated with melanoma progression. Edn3 plays varying roles during melanocyte development, promoting the proliferation and self-renewal of NC-derived multi- and bi-potential precursors as well as the survival, proliferation, differentiation and migration of committed melanocyte precursors. Melanocyte differentiation is achieved via the interaction of Ednrb and Kit signaling, with Ednrb being specifically required in the final differentiation step, rather than in the initial specification of melanocytic fate. Ednrb has also been implicated in the de-differentiation of mature melanocytes, a process that takes place during the malignant transformation of these cells. Ednrb was found to be upregulated in melanoma metastases and was shown to alter tumor–host interactions leading to melanoma progression. Antagonists to this receptor were shown to inhibit melanoma cell growth and increase the apoptotic rate of these cells, and to lead to disease stabilization in melanoma patients. Thus, Edn signaling inhibition may prove useful in the treatment of certain types of melanoma.     

Hearing dysfunction in heterozygous MitfMi‐wh/+ mice,a model for Waardenburg syndrome type 2 and Tietz syndrome

The human deafness‐pigmentation syndromes, Waardenburg syndrome (WS) type 2a, and Tietz syndrome are characterized by profound deafness but only partial cutaneous pigmentary abnormalities. Both syndromes are caused by mutations in MITF. To illuminate differences between cutaneous and otic melanocytes in these syndromes, their development and survival in heterozygous Microphthalmia‐White (Mitf^Mi‐wh/+) mice were studied and hearing function of these mice characterized. Mitf^Mi‐wh/+ mice have a profound hearing deficit, characterized by elevated auditory brainstem response thresholds, reduced distortion product otoacoustic emissions, absent endocochlear potential, loss of outer hair cells, and stria vascularis abnormalities. Mitf^Mi‐wh/+ embryos have fewer melanoblasts during embryonic development than their wild‐type littermates. Although cochlear melanocytes are present at birth, they disappear from the Mitf^Mi‐wh/+ cochlea between P1 and P7. These findings may provide insight into the mechanism of melanocyte and hearing loss in human deafness‐pigmentation syndromes such as WS and Tietz syndrome and illustrate differences between otic and follicular melanocytes.     

Genetic background strongly modifies the severity of symptoms of Hirschsprung disease, but not hearing loss in rats carrying Ednrb(sl) mutations

Hirschsprung disease (HSCR) is thought to result as a consequence of multiple gene interactions that modulate the ability of enteric neural crest cells to populate the developing gut. However, it remains unknown whether the single complete deletion of important HSCR-associated genes is sufficient to result in HSCR disease. In this study, we found that the null mutation of the Ednrb gene, thought indispensable for enteric neuron development, is insufficient to result in HSCR disease when bred onto a different genetic background in rats carrying Ednrb(sl) mutations. Moreover, we found that this mutation results in serious congenital sensorineural deafness, and these strains may be used as ideal models of Waardenburg Syndrome Type 4 (WS4). Furthermore, we evaluated how the same changed genetic background modifies three features of WS4 syndrome, aganglionosis, hearing loss, and pigment disorder in these congenic strains. We found that the same genetic background markedly changed the aganglionosis, but resulted in only slight changes to hearing loss and pigment disorder. This provided the important evidence, in support of previous studies, that different lineages of neural crest-derived cells migrating along with various pathways are regulated by different signal molecules. This study will help us to better understand complicated diseases such as HSCR and WS4 syndrome.     

EDNRB/EDN3 and Hirschsprung Disease Type II

The study of vertebrate pigmentary anomalies has greatly improved our understanding of melanocyte biology. One such disorder, Waardenburg syndrome (WS), is a mendelian trait characterized by hypopigmentation and sensorineural deafness. It is commonly subdivided into four types (WS1–4), defined by the presence or absence of additional symptoms. WS type 4 (WS4), or Shah‐Waardenburg syndrome, is also known as Hirschsprung disease Type II (HSCR II) and is characterized by an absence of epidermal melanocytes and enteric ganglia. Mutations in the genes encoding the endothelin type‐B receptor (EDNRB) and its physiological ligand endothelin 3 (EDN3) are now known to account for the majority of HSCR II patients. Null mutations in the mouse genes Ednrb and Edn3 have identified a key role for this pathway in the normal development of melanocytes and other neural crest‐derived lineages. The pleiotropic effects of genes in this pathway, on melanocyte and enteric neuron development, have been clarified by the embryologic identification of their common neural crest (NC) ancestry. EDNRB and EDN3 are transiently expressed in crest‐derived melanoblast and neuroblast precursors, and in the surrounding mesenchymal cells, respectively. The influence of EDNRB‐mediated signaling on the emigration, migration, proliferation, and differentiation of melanocyte and enteric neuron precursors, in vivo and in vitro has recently been the subject of great scrutiny. A major emergent theme is that EDN3‐induced signaling prevents the premature differentiation of melanocyte and enteric nervous system precursors and is essential between 10 and 12.5 days post‐coitum. We review the present understanding of pigment cell development in the context of EDNRB/EDN3 – a receptor‐mediated pathway with pleiotropic effects.     

Treatment of Aganglionic Megacolon Mice via Neural Stem Cell Transplantation

To explore a potential methodology for treating aganglionic megacolon, neural stem cells (NSCs) expressing engineered endothelin receptor type B (EDNRB) and glial cell-derived neurotrophic factor (GDNF) genes were transplanted into the aganglionic megacolon mice. After transplantation, the regeneration of neurons in the colon tissue was observed, and expression levels of differentiation-related genes were determined. Primary culture of NSCs was obtained from the cortex of postnatal mouse brain and infected with recombinant adenovirus expressing EDNRB and GDNF genes. The mouse model of aganglionic megacolon was developed by treating the colon tissue with 0.5 % benzalkonium chloride (BAC) to selectively remove the myenteric nerve plexus that resembles the pathological changes in the human congenital megacolon. The NSCs stably expressing the EDNRB and GDNF genes were transplanted into the benzalkonium chloride-induced mouse aganglionic colon. Survival and differentiation of the implanted stem cells were assessed after transplantation. Results showed that the EDNRB and GDNF genes were able to be expressed in primary culture of NSCs by adenovirus infection. One week after implantation, grafted NSCs survived and differentiated into neurons. Compared to the controls, elevated expression of EDNRB and GDNF was determined in BAC-induced aganglionic megacolon mice with partially improved intestinal function. Those founding indicated that the genes transfected into NSCs were expressed in vivo after transplantation. Also, this study provided favorable support for the therapeutic potential of multiple gene-modified NSC transplantation to treat Hirschsprung’s disease, a congenital disorder of the colon in which ganglion cells are absent.